Protein A and Purification of Monoclonal Antibodies
Since monoclonal antibodies (mAbs) are used for pharmaceutical applications, they are required in exceptionally high purities [A. Jungbauer, G. Carta, in: Protein Chromatography, Process Development and Scale-Up; WILEY-VCH Verlag, Weinheim (Germany) 2010].
Protein A is initially a 56 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArIS-ArIR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is originally composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family. Through these interactions in serum, where IgG molecules are bound in the wrong orientation (in relation to normal antibody function), the bacteria disrupts opsonization and phagocytosis.
Here the terms “Protein A” and “Prot A” are used interchangeably and encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a CH2/CH3 region, such as an Fc region. Protein A can be purchased commercially from Repligen, GE or Fermatech. Protein A is generally immobilized on a chromatography matrix. A functional derivative, fragment or variant of Protein A used in the methods and systems according to the present invention may be characterized by a binding constant of at K=108 M, and preferably K=109 M, for the Fc region of mouse IgG2a or human IgGI. An interaction compliant with such value for the binding constant is termed “high affinity binding” in the present context. In some embodiments, such functional derivative or variant of Protein A comprises at least part of a functional IgG binding domain of wild-type Protein A, selected from the natural domains E, D, A, B, C or engineered mutants thereof which have retained IgG binding functionality.
Also, Protein A derivatives or variants engineered to allow a single-point attachment to a solid support may also be used in the affinity chromatography step in the claimed methods.
Single point attachment generally means that the protein moiety is attached via a single covalent bond to a chromatographic support material of the Protein A affinity chromatography. Such single-point attachment may also occur by use of suitably reactive residues which are placed at an exposed amino acid position, namely in a loop, close to the N- or C-terminus or elsewhere on the outer circumference of the protein fold. Suitable reactive groups are e.g. sulfhydryl or amino functions.
In some embodiments, Protein A derivatives of variants are attached via multi-point attachment to suitable a chromatography matrix.
In general, biotechnological processes only provide highly impure mAbs in very low concentrations so that the removal of these impurities needs a set of complex isolation and purification steps, also referred to as downstream process. The efficiency of the downstream process affects the manufacturing costs of mAbs significantly, which results in the continuous ambition to improve the procedure of every sequential step [R. Freitag and C. Horváth; Adv. Biochem. Eng./Biotechnol., 1996, 53, 17-59]. Of all the processes steps involved, protein A chromatography (also referred to as affinity chromatography) is, invariably, the most crucial and expensive step. This means that Protein A affinity chromatography is one of the most crucial purification steps in the downstream processing of monoclonal antibodies mAbs.
In detail, the crude multi-component solution passes through a column packed with a stationary phase comprising immobilized protein A on a solid porous support. The desired mAb is captured by specific interactions between mAb and protein A while the impurities leave the column together with the leaving solvent. The captured mAb is recovered by usage of an appropriate eluant [P. Cuatrecasas, M. Wilchek, C. B. Anfinsen, Proc. Natl. Acad. Sci. USA, 61, 636 (1968)].
In practice, this bind and elute operation consists of a sequence of several steps. In the first step the column is washed with the buffer in which the target molecule containing feed-stream will be loaded. In the next step, feed stream, containing target mAb and impurities, is passed through the column containing protein A stationary phase. In this step the target mAb molecule is captured by the virtue of its specific affinity towards protein A, while the impurities mostly pass through. Subsequently the stationary protein A phase is flushed with the washing buffer to remove remaining impurities. Then, the captured mAb molecules are recovered (eluted) by passing elution buffer through the column. The elution of mAb is caused by the chemical environment generated by the elution buffer, which induces a shift in affinity between target mAb molecule and protein A. The column is finally cleaned and regenerated for repeat bind and elute of mAb for several more cycles.
Thus, affinity chromatography relies on very specific bonding interactions between target mAb and the surface of stationary phase. Ideally this specific binding should occur in such a way that every component of the starting mixture without substantial affinity to the surface passes through the chromatographic column, while the desirable molecule is retained. Various physiochemical interactions including electrostatic, hydrophobic, van der Waals and hydrogen bonding, the nature of the medium carrying the starting mixture, the complementary arrangement of the target molecule and the binding sites on the surface of the resin are typically responsible for a desired biospecifity [K. Huse, H.-J. Böhme and G. H. Scholz, J. Biochem. Biophys. Methods, 2002, 51, 217]. However, the bond between surface and biomolecule needs to be weak enough for elution by changing pH or salt concentration, or by addition of competitive inhibitor in solution [A. Jungbauer G. Carta, Protein Chromatography, WILEY-VCH Verlag, 2010; R. Freitag and C. Horváth, Adv. Biochem. Eng./Biotechnol., 1996, 53, 17; A. A. Shukla, B. Hubbard, T. Tressel, S. Guhan and D. Low, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci., 2007, 848, 28; P. Cuatrecasas, M. Wilchek and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A., 1968, 61, 636]. Obviously, efficiency and performance of the affinity chromatography depends on the described specific interactions of target molecules to the surface. However, due to the wide variety of mAbs produced, it is necessary that the design of stationary phases is evolved to the needs of specific mAb. Generally, such a design is achieved by modifying stationary phases with the ligand that possesses specific affinity for the mAb under consideration. In addition to the specific interactions, choice of a protein A chromatography medium depends on several further process parameters, such as strength (typically characterized by the pressure response to the applied flow rates), and stability to several solutions used in all the steps of protein A chromatography.
In summary, design of an efficient affinity chromatography medium requires judicious choice of the ligand, the stationary phase and strategy for attachment of ligand to the stationary phase. The choice of all these elements is governed by the specificity and efficiency in purifying mAbs, stability of the stationary phase as a whole, and ultimately, the cost of the affinity medium. Most ligands are suitable due to their natural affinity to the desirable molecule, e.g. antibodies, antigens, lectins, receptors, enzyme inhibitors, hormones or biomimetic ligands [R. Freitag and C. Horváth, Adv. Biochem. Eng./Biotechnol., 1996, 53, 17; K. Huse, H.-J. Böhme and G. H. Scholz, J. Biochem. Biophys. Methods, 2002, 51, 217]. Protein A is a successfully applied and well proved affinity ligand for the capture of monoclonal immunoglobulin G antibodies. [R. Freitag and C. Horváth, Adv. Biochem. Eng./Biotechnol., 1996, 53, 17; A. A. Shukla, B. Hubbard, T. Tressel, S. Guhan and D. Low, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci., 2007, 848, 28; S. R. Narayanan, J. Chromatogr., A, 1994, 658, 237; K. Huse, H.-J. Böhme and G. H. Scholz, J. Biochem. Biophys. Methods, 2002, 51, 217; R. Hahn, P. Bauerhansl, K. Shimahara, C. Wizniewski, A. Tscheliessnig and A. Jungbauer, J. Chromatogr., A, 2005, 1093, 98]. While protein A imparts the specificity to the medium, the mechanical stability is primarily provided by the underlying support material. Several materials are used as a supporting material for protein A stationary phase. In addition to the mechanical strength, the supporting materials should also be able to provide high surface area to attach protein A in efficient fashion. Thus, a matrix is necessary which satisfies these claims and which is preferably mechanically and chemically stable.
Furthermore, an important feature of chromatographic columns is their life time. Affinity chromatography for industrial applications consists of four stages: adsorption, washing, elution, regeneration [S. R. Narayanan, J. Chromatogr., A, 1994, 658, 237]. So, performance of the columns is expected to be constant over the range of numerous cycles to guarantee the reproducibility of the process.
The high specifically bindings of mAb to immobilized protein A in affinity chromatography for purification of mAb is caused by interactions with the fragmental crystallisable (FC) regions of mAb and appropriate sites of protein A. Protein A originates from the bacteria Staphylococcus aureus and has actually the purpose to protect the bacteria from mammal's immune systems while binding IgG on a way that makes it inoperative. This skill is basically utilized, using protein A as affinity chromatography ligand. Protein A has five structurally related, homologous domains which can all bind to respective FC region of IgG [K. Huse, H.-J. Böhme and G. H. Scholz, J. Biochem. Biophys. Methods, 2002, 51, 217; S. Ibrahim, Scand. J. Immunol., 1993, 38, 368; S. Hober, K. Nord and M. Linhult, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci., 2007, 848, 40]. Proteins are macromolecules and their activity is based on tertiary structures (e.g. FC region) so that their orientation in space and conditions like the media's pH are important influencing parameters for successfully complex building between ligand and target molecule—in this case between protein A and IgG. But the requirement of an appropriate, specific binding environment is on the other hand the key for the detachment during elution. The complex building of IgG FC region with protein A requires an appropriate orientation in pores. The 3D view and the arrangement of IgG-protA-complex are worked out by H. Yang et al. [H. Yang, P. V. Gurgel, D. K. Williams, Jr., J. Cavanagh, D. C. Muddiman and R. G. Carbonell, J. Mol. Recognit., 2010, 23, 27].
The difference in size dimensions is based on the molecular weight of both proteins, IgG (144 kDa) is significant bigger than protein A (40-60 kDa). Those facts need to be considered for pore structure design of supporting material.
Besides specific binding sites for mAb, protein A need to provide functional groups to achieve its immobilization on support material surface, keeping accessibility of active sites. Established handles for immobilization of protein A are amine groups and thiol groups which can be covalently attached to the stationary phase equipped with complementary reactive groups such as epoxy, carboxylic acid, or aldehyde, for example [H. Ahmed, Principles and Reactions of Protein Extraction, Purification, and Characterization, CRC Press LLC, 2004; P. Cuatrecasas, J. Biol. Chem., 1970, 245, 3059; Z. Pan, H. Zou, W. Mo, X. Huang and R. Wu, Anal. Chim. Acta, 2002, 466, 141]. There are wild type protein A equipped with suitable anchor groups, but alteration of protein A by mutation allows the creation of tailor made protein A molecules concerning the coupling chemistry, the performance and the alkaline resistance, attributable to the variation of amino acid residues. The availability of diverse protein A in regard to functional groups promotes the consideration of different immobilization strategies. One-point or multiple-point attachments, the choice of support material, and chemical surface activation dependent on functional groups of protein A are variables.
Multiple-point attachment compared with one-point attachment, for example, promises an improvement of durability of protein A immobilization to prevent any protein A leaching during mAb purification.
Thiols are generally more reactive than amines due to their stronger nucleophilic nature so that thiol containing protein A can be immobilized very efficiently on epoxy groups providing surfaces. There are also several coupling methods for ligands containing primary amines.
Focus of the current work is the application of protein A with multiple terminal amines for the immobilization on activated porous metal oxide particles.
Stationary Phase
While the specificity of interactions between mAb and surface is dependent on the PrA ligand, the porous matrices on which the protein A ligands are immobilized need to satisfy certain criteria. Membranes and monoliths have also been considered as supports for protein chromatography, but porous, micron size beads are typically the most popular and widely used choice for support materials [A. Jungbauer, G. Carta, in: Protein Chromatography, Process Development and Scale-Up; WILEY-VCH Verlag, Weinheim (Germany) 2010]. The efficiency of the protein A medium is generally measured in terms of the binding capacity (in static or dynamic mode) per unit volume of the resin. The binding capacity is a function of the physical attributes of the porous beads, such as surface area, pore size, pore volume, and bead size. However in general, increasing the surface area provides more functional groups per unit volume for attachment of protein A, and in turn, can increase the binding capacity. However, increasing surface area may come at the cost of mechanical stability of the porous beads packed in a column.
The mechanically stability is necessary for the porous beads to withstand the operating flow rates and resulting pressure, because it is economical to operate at high flow rates as compared to low flow rates at the same dynamic binding capacity. However, such high rates cause dense packing of porous beads in the columns, and high pressure drops. From this point of view, it is desirable to use a material that has uniform particle size and narrow pore size distribution, and therefore, allows accurate packing. Particles with small pore diameter usually provide high surface areas. However, the pore size needs to be sufficient to allow diffusion of the biomolecules. Beside these physical attributes, the support material should also possess several chemical attributes. Because protein A chromatography operates on the principle of selective affinity between the desired molecule and the stationary phase, the stationary phase should ideally lack any potential of interaction (typically referred to as non-specific adsorptive interaction or binding) with the desired molecule. [A. Jungbauer, G. Carta, in: Protein Chromatography, Process Development and Scale-Up; WILEY-VCH Verlag, Weinheim (Germany) 2010]
In addition to the inherent needs, based on separation performance, recently, there is demand that the columns are regenerated (“cleaned”) using aqueous sodium hydroxide solution. Such a practice, typically referred to as “clean-in-place” (CIP), requires that the support material should offer chemical resistance towards the high pH alkaline conditions [M. Rogers, M. Hiraoka-Sutow, P. Mak, F. Mann and B. Lebreton, J. Chromatogr., A, 2009, 1216, 4589].
With these criteria in mind, there are several supporting materials which are widely used in practice, including natural carbohydrate polymers, synthetic polymers, and inorganic materials. Natural carbohydrate polymers include agarose, cellulose, dextran and chitosan, are commercially available for protein chromatography application [A. Jungbauer, G. Carta, in: Protein Chromatography, Process Development and Scale-Up; WILEY-VCH Verlag, Weinheim (Germany) 2010].
First descriptions of the use of modified natural carbohydrate polymer for ion exchange date back to the 1950s [E. A. Peterson and H. A. Sober, J. Am. Chem. Soc., 1956, 78, 75131]. Typically, a porous agarose gel can be prepared by cooling a hot agarose solution. Primary feature of those materials is their hydrophilic character (low non-specific adsorption) and the accessibility to a high amount of hydroxyl groups for chemical modification and ligand immobilization so that high capacities are reachable. Additionally, carbohydrates are chemically resistant at extreme alkaline conditions, and therefore suitable for CIP.
However, because of low solid density, these agarose-based materials lack mechanical stability restricting their use at very high flow rates and pressure. The mechanical stability of these materials can be improved by chemical crosslinking. However, such chemical crosslinking typically use the hydroxyl groups as handles, limiting the extent to which the agarose gel can be crosslinked without sacrificing the hydroxyl groups available as handles for further ligand immobilization. As a result, the mechanical strength of these crosslinked agarose gels may not match competitive materials like inorganic materials.
Porous synthetic polymer beads are also used as stationary phase for affinity chromatography.
These synthetic polymer beads of desired particle sizes are usually prepared by suspension or emulsion polymerization using a judiciously chosen porogen, which also yields high surface areas. This suspension polymerization typically involves seeding a radical polymerization of a monomer with porogen, and has been proven to yield narrow distribution of particle sizes [T. Ellingsen, O. Aune, J. Ugelstad and S. Hagen, J. Chromatogr., 1990, 535, 147]. However, due to the nature of the synthesis of beads, and depending on the monomer used for the synthesis, synthetic polymers can be significantly more hydrophobic than natural carbohydrate polymers. Depending on the choice of monomer, polymer beads can also provide a high density of functional groups, available for surface modification, thus, hydrophilic surfaces can be obtained. Regardless, due to the commercial availability of large number of functional monomers, the polymer beads generated via suspension methods can be chemically diverse and highly tunable. Commonly used polymers are polyacrylamides and polyacrylates. 2-Hydroxyethyl methacrylate (HEMA) is an example for a high hydrophilic and biocompatible polymer [A. Jungbauer, G. Carta, in: Protein Chromatography, Process Development and Scale-Up; WILEY-VCH Verlag, Weinheim (Germany) 2010]. Due to their higher solid density those materials are usually more mechanically stable than carbohydrates, but they tend to swell in aqueous media the more hydrophilic they are. One more benefit is the high chemical resistance of polymer beads so that the CIP is expectably not affecting the performance of columns packed with polymer beads caused by damage of the resin structure.
A third class of supporting materials is inorganic, or ceramic, particles. Particles of that class are for example so called controlled pore glass (CPG) particles. FIG. 1 shows such particles and their pore structure.
Silica based beads, such as CPG as commercialized by Merck Millipore, have the highest solid density, and offer excellent mechanical strength. Especially the use of CPG with its large, interconnected and uniform pores enables excellent flow properties [Schnabel, R.; Langer, P., J. Chromatogr., 1991, 544, 137]. Moreover, a good mass transfer due to the morphology allows a quite unhindered pore diffusion of proteins so that these properties benefit higher scales. Maintaining of shape and excellent flow properties promote high flow rates with linear pressure increasing whereas the unhindered mass transfer allows short retention times. However, CIP is the main limitation in using inorganic supporting materials such as CPG [M. Rogers, M. Hiraoka-Sutow, P. Mak, F. Mann and B. Lebreton, J. Chromatogr., A, 2009, 1216, 4589]. Usually they are based on SiO2 which is soluble in strongly alkaline media so that the lifetime of those columns is limited in the number of process cycles. Furthermore, higher non-specific bindings compared to agarose based materials are observed as well as the surface modification on inorganic supports is in general more challenging. Nonetheless protein chromatography media based on inorganic materials has been successfully established in this industrial setting where CIP with sodium hydroxide is not practiced.
Overall, each of the base matrices provides one or more advantages for protein A affinity media, but the most desirable improvement is in the caustic stability of CPG.
This means, for the use of porous inorganic particles, like CPG, with all its benefits in high scale protein A chromatography, the enhancement of the alkaline resistance is desirable. Several methods concerning this issue for silica based materials are described in literature. Many of them include procedures which are referred to as “end capping” reactions. Residual silanols are treated with chlorosilanes to shield the silica surface. This technique enhances the alkaline resistance but the lifetime is still not satisfying for high pH treatment, further it is more suitable in reversed phase chromatography application [Nawrocki, J., Dunlap, C., McCormick, A., Carr, P. W., J. Chromatogr., A, 2004, 1028, 1; Samuelsson, J. A., Franz, Stanley, B. J., Fornstedt, T., J. Chromatogr., A, 2007, 1163, 177; Kirkland, J. J., van Straten, M. A., Claessens, H. A., J. Chromatogr., A, 1995, 691, 3; Kirkland, J. J., van Straten, M. A., Claessens, H. A., J. Chromatogr., A, 1996, 728, 259].
Polymer coating on silica surfaces is a further option which is well investigated for plenty of polymers [Petro, D. Berek, M., Chromatographia, 1993, 37, 549 36]. For protein purification applications, significant enhancement of alkaline resistance of silica gel has been described after coating the surface with carbohydrate based polymers such as dextran or chitosan. Afterwards the polymeric surface is opened for commonly used modification strategies [Fengna Xi and Jianmin Wu, React. Funct. Polym., 2006, 66, 682; E. Boschetti, P. Girot and L. Guerrier, J. Chromatogr., 1990, 523, 35]. In addition to these investigations J. Nawrocki et al. [J. Chromatogr., A, 2004, 1028, 1] compared the alkaline resistance of silica and various metal oxide based packings in an extensive report, as well as further important properties for chromatographic application.
Considering all these investigations, for immobilization of protein A onto the surface of porous beads, it is necessary that the surface offers a reactive handle that is suitable for functional groups of protein A. Obviously, protein A needs to possess the reactive functional groups without much adulteration in its structure and property.